Efficient antigen presentation is essential to induce effective cellular and humoral immune responses. Thus, one central goal of immunotherapy and vaccine development is to enhance antigen presentation to induce potent and broad immune responses. FcyR-mediated internalization can activate DCs as well as enhance antigen presentation up to 10,000 times more efficiently than the fluid phase pinocytosis. The hypothesis of this application is that DCs can be transduced to produce and secrete a fusion protein consisting of an antigen and a cell-binding domain, which, in addition to inducing B-cell responses, would be efficiently captured by receptor-mediated internalization and processed in the endosomal pathway by DCs for MHC-II as well as -I presentation to induce helper and cytotoxic T-cells. In the preliminary study, a model hepatitis B virus e antigen (HBeAg) was fused with an IgG Fc fragment, and superior antigen-specific CD4+ helper and CD8+ cytotoxic T-cell, as well as, B-cell responses were induced by the DCs expressing the HBe-Fc fusion protein, termed "retrogen." This application is aimed to further develop this retrogen strategy that uses a unifying antigen presentation mechanism to efficiently induce all arms of the adaptive immunity for immunotherapy and vaccine development. In Aim 1, it will be assessed if the retrogen strategy can enhance antigen uptake by DCs via receptor-mediated internalization, and if the retrogens can activate DCs as well. In Aim 2, the mechanisms of retrogen enhancement of helper and CTLs responses will be dissected to optimize the retrogen design by generating and assessing retrogen mutants in MHC-II or FcyRs knock-out and wild type mice. The ability of other cell-binding domains to enhance immune responses will be determined and compared to their potency with HBe-Fc in mice, In Aim 3, the applicability of retrogen strategy to enhance immunogenicity of other antigens such as the human papillomavirus (HPV)-E7 antigen in vitro and in vivo will be tested. Finally, it will be tested whether this retrogen strategy can be incorporated into DNA vaccines to enhance immune responses. This study will contribute to establishing the novel retrogen strategy that is capable of inducing T helper, cytotoxic killer, and B-cell responses. This retrogen strategy may provide a powerful and generic means for the development of vaccines and immunotherapy.